High-grade gliomas are the leading cause of brain tumor-related death in both children and adults. We have recently shown that cortical neuronal activity promotes the growth of high-grade glioma in vitro and in vivo. A major mechanism mediating the growth-promoting effect of neuronal activity on high grade glioma is activity-regulated secretion of the synaptic protein neuroligin-3 (NLGN3) ectodomain, which we have shown to be both sufficient and necessary in situ and in vitro. Soluble neuroligin-3 stimulates the PI3K-mTOR pathway in glioma cells and also induces feed-forward expression of glioma NLGN3, but the upstream signaling events are not yet elucidated. In Aim 1 of this proposal, we now seek to confirm the necessity of NLGN3 for in vivo glioma growth and to determine the cellular origin(s) of secreted NLGN3 using constitutive and inducible NLGN3 genetic deletion strategies together with in vivo optogenetic techniques and patient-derived orthotopic xenograft models. In Aim 2 we will identify the activity-regulated enzyme responsible for NLGN3 secretion using pharmacological and genetic strategies. In Aim 3 we will identify the NLGN3 binding partner in glioma cells using the range of adult and pediatric patient-derived high grade glioma models we have developed, co-immunoprecipitation and mass spectrometric analysis. The proposed experiments will deepen our understanding of the mechanisms by which neurons promote cancer growth in the glioma microenvironment and will identify potential targets of therapy for these deadly cancers.